Conjugate containing an antigenic peptide and its use for preventing bacterial infections caused by shigella

Abstract

The invention relates to a conjugate composed of an antigenic peptide presenting an epitope of the Shigella flexneri 3a OmpC protein of SEQ ID NO: 1, conjugated via the C- terminal residue of the peptide to a human serum albumin (MSA) carrier protein molecule, a vaccine composition containing said conjugate, and its use for preventing bacterial infections caused by Shigella bacteria.

Description

[0001] Conjugate Containing an Antigenic Peptide and Its Use for Preventing Bacterial Infections Caused by Shigella

[0002] The invention relates to a conjugate containing peptide antigens and a carrier protein, a vaccine composition containing conjugate, and its use for preventing bacterial infections caused by bacteria from the genus Shigella.

[0003] Bacterial foodborne infections caused by enterobacteria of the Shigella genus directly cause bloody, watery diarrhea, often accompanied by severe abdominal pain, nausea, and vomiting, and are referred to bacillary dysentery or dysentery. Shigelosis (dysentery, bacillary dysentery) is a disease characterized by acute inflammation of the gastrointestinal tract caused by the invasion of Shigella bacteria into the epithelial cells lining the intestinal walls. The most characteristic symptoms of shigellosis include watery, bloody, or mucous diarrhea, nausea, vomiting, fever, and abdominal pain (Ram PK, Crump JA, Gupta SK, Miller MA, Mintz ED: Part II. Analysis of data gaps pertaining to Shigella infections in low and medium human development index countries, 1984-2005. Epidemiol. Infect., 2008; 136: 577-603). Bacterial foodborne infections caused by enterobacteria of the Shigella genus can be treated with antibiotics. However, it should be emphasized that in recent years, many antibiotic-resistant strains have emerged among bacteria belonging to the Shigella genus. Some strains even exhibit multidrug resistance. The increasing number of antibiotic-resistant bacterial strains is currently one of the most important problems, effectively hindering the treatment of foodborne infections (Livermore DM, Brown DF: Detection of beta-lactamase-mediated resistance. J. Antimicrob. Chemother., 2001, 48: 59-64; Urvashi, Sonal Saxena and Renu Dutta: Antimicrobial Resistance Pattern of Shigella species Over Five Years at a Tertiarycare Teaching Hospital in North India. J Health Popul Nutr. 2011 June; 29(3): 292-295). Local outbreaks of Shigella infections are increasingly reported, and the resistance of these strains has been gradually increasing since 2013. Slowly, most cheap oral antibiotics, such as ampicillin and trimethoprim, are becoming ineffective. Currently, the drugs of choice are ciprofloxacin and azithromycin. However, approximately 88,000 Shigella cases in the United States demonstrate drug resistance (U.S. Centers for Disease Control and Prevention. Antibiotic resistance threats in the United States. 2019. Available online:

[0004]

[0005] (accessed on October 21 , 2021 )).

[0006] These infections are also difficult to diagnose due to the fact that the bacteria causing dysentery are antigenically and morphologically very similar to E. col / species. Therefore, it is difficult to distinguish them not only from pathogenic E. coli strains, but also from strains naturally occurring in the human body as commensal flora. According to the WHO, almost one million people die from shigellosis worldwide each year. These poisonings are dangerous not only for children and the elderly, among whom the mortality rate is highest,but also for people whose immune systems demonstrate immunodeficiencies or are immunosuppressed due to cytostatic treatment and are therefore unable to resist pathogens. Furthermore, the morbidity rate among travellers to developing countries is very high (it is estimated that approximately 50% of the population traveling to countries with poor sanitary conditions is affected by so-called "traveller’s diarrhea"). Shigella poses a serious threat to public health due to its resistance to many antibiotics, lengthy diagnostic procedures, and the unavailability of a licensed vaccine. Research on a vaccine that induces a long-lasting immune response against Shigella strains is being conducted in many research institutions worldwide. Despite the efforts of many research centres, such a vaccine is still not available, and its development would be a major achievement not only in Poland but also internationally, where foodborne infections pose a serious threat. This demonstrates the high demand for this type of vaccine, but also the fact that selecting and characterizing such an antigen is not an easy task. Currently, no vaccine protecting against bacillary dysentery is available on the pharmaceutical market. Most vaccine preparation strategies against bacteria causing foodborne illnesses are based on the use of whole, killed or live-attenuated bacterial strains, as well as whole cell antigens. Currently, one of the most popular and considered solutions are strains attenuated (Simon JK, Maciel M Jr, Weld ED, Wahid R, Pasetti MF, Picking WL, Kotloff KL, Levine MM, Sztein MB: Antigenspecific IgA B memory cell responses to Shigella antigens elicited in volunteers immunized with live attenuated Shigella flexneri 2a oral vaccine candidates. Clin Immunol. 2011,139:185-92; Raqib R, Sarker P, Zaman K, Alam NH, Wierzba TF, Maier N, Talukder K, Baqui AH, Suvarnapunya AE, Qadri F et al. A phase I trial of WRSS1, a Shigella sonnei live oral vaccine in Bangladeshi adults and children. Hum. Vaccine Immunother.2019, 15, 1326-1337), killed (Chakraborty S, Harro C, DeNearing B, Bream J, Bauers N, Dally L, Flores J, Van de Verg L, Sack DA, Walker R: Evaluation of the Safety, Tolerability, and Immunogenicity of an Oral, Inactivated Whole-Cell Shigella flexneri 2a Vaccine in Healthy Adult Subjects. Clin. Vaccine Immunol.2016, 23, 315-325), or subunit vaccines, which most often consist of lipopolysaccharides and bacterial proteins (Tribble D, Kaminski R, Cantrell J, Nelson M, Porter C, Baqar S, Williams C, Arora R, Saunders J, Ananthakrishnan M, Sanders J, Zaucha G, Turbyfill R, Oaks E: Safety and Immunogenicity of a Shigella flexneri 2a Invaplex 50 intranasal vaccine in adult volunteers. Vaccine, 2010; 28: 6076-6085) or polysaccharide antigens combined with carrier proteins (Talaat KR, Alaimo C, Martin P, Bourgeois AL, Dreyer AM, Kaminski RW et al. Human challenge study with a Shigella bioconjugate vaccine: Analyses of clinical efficacy and correlates of protection. EBioMedicine.2021 Apr;66. 103310). Some vaccine prototypes are or have been tested in Phase 1 and 2 clinical trials, demonstrating their safety and immunogenicity. Several of them have been tested in human infection models using a virulent Shigella strain (Talaat KR, Alaimo C, Martin P, Bourgeois AL, Dreyer AM, Kaminski RW et al. Human challenge study with a Shigella bioconjugate vaccine:Analyses of clinical efficacy and correlates of protection. EBioMedicine. 2021 Apr;66.

[0007] 103310).

[0008] According to the latest knowledge, the most advanced vaccine is the recombinant conjugate vaccine administered parenterally. This is a monovalent synthetic vaccine SF2a-TT15 (NCT04602975) with S. flexneri 2a, currently being tested in a group of volunteers and demonstrating immunogenic properties (Cohen D, Atsmon J, Artaud C, Meron-Sudai S, Gougeon ML, Bialik A, Goren S, Asato V, Ariel-Cohen O, Reizis A et al.: Safety and immunogenicity of a synthetic carbohydrate conjugate vaccine against Shigella flexneri 2a in healthy adult volunteers: A phase 1, dose-escalating, single-blind, randomized, placebo-controlled study. Lancet Infect. Dis. 2021, 21, 546-55). Another advanced prototype is the multivalent vaccine altSonflex1-2-3 (NCT05073003), whose monovalent S. sonnei precursor was safe and immunogenic in human volunteers, but proved ineffective in protecting against infection (Frenck RW, Conti V, Ferruzzi P, Ndiaye AGW, Parker S, McNeal MM, Dickey M, Granada JP, Cilio GL, de Ryck I et al.: Efficacy, safety, and immunogenicity of the Shigella sonnei 1790GAHB GMMA candidate vaccine: Results from a phase 2b randomized, placebo-controlled challenge study in adults. EClinicalMedicine 2021, 39, 1010768). The multivalent S4V conjugate (NCT04056117) has also been shown to be safe, immunogenic, and its efficacy is currently being studied (Talaat KR, Alaimo C, Martin P, Bourgeois AL, Dreyer AM, Kaminski RW et al. Human challenge study with a Shigella bioconjugate vaccine: Analyses of clinical efficacy and correlates of protection. EBioMedicine.2021 Apr;66. 103310). All three of these vaccines are currently being tested in pediatric populations in Kenya.

[0009] Many teams are focused on research related to attenuated vaccines, some of which are in the first phase of clinical trials. A weakened strain of Shigella flexneri 2a SC602 causes elevation in serum volunteers the level of IgG antibodies directed against bacteria lipopolysaccharides (Rahman KM, Arifeen SE, Zaman K, Rahman M, Raqib R, Yunus M, Begum N, Islam MS, Sohel BM, Rahman M, Venkatesan M, Hale TL, Isenbarger DW, Sansonetti PJ, Black RE, Baqui AH: Safety, dose, immunogenicity, and transmissibility of an oral live attenuated Shigella flexneri 2a vaccine candidate (SC602) among healthy adults and school children in Matlab, Bangladesh. Vaccine. 2011 Feb 1 ;29(6):1347-54). However, when using this type of vaccine, there is a risk of mutation and reversion of the weakened strain to a fully virulent form. Furthermore, these vaccines are very laborious to produce and difficult to store. Regarding the development of subunit vaccines, the most advanced so far is the Invaplex complex vaccine, based on protein antigens (IpaA and IpaB proteins) and lipopolysaccharides isolated from the Shigella flexneri 2a strain. Studies currently in the preclinical phase have shown that administration of the vaccine increases the levels of IgG and IgA antibodies in the serum of volunteers. Furthermore, aside from short-term irritation of the nasal mucosa, no other side effects were observed in vaccinated volunteers. Although the preliminary results obtained for the Invaplex 50 vaccine are promising, its economic production is quite expensive, primarily due to thecomplex process of obtaining the individual components, as well as standardizing its composition after isolation from bacterial material. Furthermore, endotoxin (LPS) is a serious problem with this type of vaccine, which can cause poisoning in the event of an overdose. Due to the high cost of obtaining this vaccine, work is currently underway on a synthetic equivalent containing antigens originally isolated from bacterial strains (Tribble D, Kaminski R, Cantrell J, Nelson M, Porter C, Baqar S, Williams C, Arora R, Saunders J, Ananthakrishnan M, Sanders J, Zaucha G, Turbyfill R, Oaks E: Safety and immunogenicity of a Shigella flexneri 2a Invaplex 50 intranasal vaccine in adult volunteers. Vaccine, 2010; 28: 6076-6085; Turbyfill KR, Clarkson KA, Oaks EV, Kaminski RW. From Concept to Clinical Product: A Brief History of the Novel Shigella Invaplex Vaccine’s Refinement and Evolution. Vaccines (Basel).2022 Apr 1 ;10(4):548).

[0010] Document WO2021255684 discloses a quadrivalent bioconjugate vaccine, describing Shigella O-polysaccharide antigens from Shigella flexneri serotypes 2a, 3a, 6 and Shigella sonnei covalently linked to a protein carrier. Document WO2021236327 discloses a vector vaccine: a recombinant plasmid of minimally invasive Shigella constructing pRISM and pRISM-G, and a method for inducing an immune response to Shigella in test subjects, the method comprising administering the composition in a dose sufficient to induce an immune response to Shigella in test subjects. Document PL220297B1 discloses peptides suitable for vaccine construction to induce protection against Enterobacteriaceae, particularly bacteria of the Shigella genus, particularly in cases of humoral immunity deficiency. The disclosed peptides constitute epitopes of the OmpC protein from Shigella flexneri 3a. Document PL235826 discloses conjugates of the OmpC protein epitope with various carriers, e.g., tetanus toxoid, diphtheria toxoid, or bovine serum albumin. Unfortunately, direct peptide-carrier protein conjugations have led to the creation of molecules with poor immunogenicity.

[0011] Previously proposed attenuated vaccines carry a significant risk associated with the possibility of reversion of weakened strains and reversion to their pathogenicity, or generate significant costs associated with the isolation and purification of natural bacterial antigens. These vaccines may also be associated with the risk of a number of side reactions, including allergies caused by the presence of many components of bacterial cells. Furthermore, vaccines containing live or weakened strains cannot be administered to individuals with acquired immunodeficiency syndrome (e.g., AIDS patients) or to patients with temporary immune deficiency, such as those with various types of cancer or undergoing chemotherapy.

[0012] The problems associated with the use of whole bacterial cells in vaccines do not apply to new-generation vaccines, which contain only a set of selected antigens that elicit a specific immune response, significantly reducing the occurrence of side effects.

[0013] Therefore, there is a need in the current state of the art to develop a cheap, safe, and effective vaccine that would contribute to a decrease in morbidity and mortality, as wellas to reduce treatment costs, particularly hospitalization. The aim of the invention is to provide a vaccine containing a unique antigen present in most pathogenic bacteria and inducing a response in the host's immune system, which offers a real chance of reducing problems associated with foodborne infections caused by strains of the Shigella genus. The main recipients of such a vaccine would be people living in countries with poor sanitary conditions. However, such a vaccine would also be a valuable product for tourists and travellers to these countries, as well as military personnel on missions. The invention provides a conjugate composed of an antigenic peptide presenting an epitope of the Shigella flexneri 3a OmpC protein of SEQ ID NO: 1 , conjugated via the C-terminal residue of the peptide to a human serum albumin (HSA) carrier protein molecule. The invention also provides a vaccine composition containing the aforementioned conjugate and an adjuvant.

[0014] The invention also provides the aforementioned conjugate for use in preventing bacterial infections caused by Shigella bacteria.

[0015] Preferably, the bacterial infection is bacillary dysentery.

[0016] The conjugate of the invention comprises the peptide GLNRYDERYIGGGGGGC (SEQ ID NO: 1) and the HSA carrier protein: GLNRYDERYIGGGGGGC - HSA. Instead of O-polysaccharide antigens, the vaccine according to the invention is based on a peptide antigen, making it more universal and potentially protective against a wider range of serotypes.

[0017] The peptide itself, although crucial in inducing a specific immune response, is a hapten and it is too small to be properly presented to the immune system. Furthermore, the free peptide undergoes proteolysis very quickly after introduction into a living system, therefore protecting it with a suitable carrier is a key step in the construction of an effective vaccine prototype.

[0018] The vaccine according to the invention is based on a unique peptide sequence, constituting a loop of the S. flexnerii 3a membrane protein OmpC. In its natural environment, this peptide is exposed by the pathogen to the external environment and thus provides an ideal shield for the immune system. Immunization of mice with the OmpC protein protects them against the virulent strain. However, due to the difficult purification process and the expression of the OmpC protein, which loses its stability after isolation, it was decided to use only the epitopic loop as the peptide antigen for the vaccine.

[0019] A peptide vaccine with a defined amino acid sequence has a significant advantage over known solutions. A conjugate based on a peptide epitope and a neutral HSA protein occurring in humans is, above all, a safe product, carrying no risk of disease as is the case with attenuated strains. Additionally, it will act as an adjuvant, supporting the response tothe specific peptide. Furthermore, the vaccine according to the invention will not cause an autoimmune response as a whole, because it will be based on a defined antigen and the carrier of the naturally occurring HSA protein in humans (albumin), rather than a diverse mixture of antigens. This risk is always high when using vaccines based on whole, killed, or attenuated bacterial cells. Furthermore, the peptide-protein vaccine can be produced on an industrial scale while maintaining minimal production costs. For comparison, the Invaplex vaccine, based on protein antigens and lipopolysaccharides isolated from the Shigella flexneri 2a strain, despite its effectiveness, cannot be introduced into mass production due to the enormous costs associated with the isolation and purification of bacterial antigens.

[0020] It should be noted that the technology based on the conjugation of a conjugate composed of a peptide (epitope), a linker (GGGGG), and the HSA protein offers low-cost scalability and stable and reproducible production, resulting in the safety of the final product.

[0021] A vaccine according to the invention, produced entirely using protein engineering tools in a ready-to-administer form, would be a highly reproducible molecule, easy to characterize and obtain, and therefore inexpensive to produce. The vaccine according to the invention is based on a peptide antigen (constituting an epitope recognized by antibodies in human cord blood), for which a linker was designed and an ideal carrier was selected, which will enable very good loading of the peptides and proper presentation of the antigen to the immune system.

[0022] The solution according to the invention, concerning the construction of a peptide vaccine against pathogenic Shigella strains, is innovative. It departs from traditional methods of obtaining attenuated vaccines or those using whole cellular antigens. Therefore, it excludes the possibility of infections due to reversion of weakened strains and significantly reduces the costs associated with vaccine production and storage. The invention uses only those parts of the antigen that have a real impact on the development of the immune response. The synthesis of short peptides representing surface epitopes of outer membrane proteins of pathogenic bacteria that elicit an immune response not only eliminates the risk of infection during vaccination but also significantly reduces the cost of obtaining the vaccine.

[0023] A peptide sequence mimicking an antigenic determinant present on the surface of a bacterial cell is an ideal antigen for constructing a peptide vaccine. The main advantages of peptide vaccines are safety and the absence of the risk of post-vaccination infection. Furthermore, such vaccines are relatively inexpensive to produce, and their chemical composition is easy to determine. These vaccines are free of components such as LPS or toxins, which cause additional inflammation and excessive reactogenicity of the preparation, and in critical cases, toxicity. An undoubted advantage of using peptide antigens is the use of a well-defined and standardized component, with a structure characteristic of a native protein antigen. Such a vaccine can be easily modifiedchemically, which can be crucial in the event of an allergy or the induction of an autoimmune response directed against one's own tissues. By chemically conjugating peptides to molecules such as lipids, sugars, or phosphate groups, we can change the properties of the vaccine, influencing its immunogenicity, stability, and solubility. Furthermore, it is possible to create polyvalent vaccines in which many different antigens are conjugated to a single carrier molecule. The use of a conjugate for immunization allows for the replacement of a classic vaccine based on thermally inactivated bacteria with a vaccine based on synthetic fragments representing the main bacterial cell surface antigen isolated from the OmpC protein. Unlike classic vaccines, the synthetic vaccine of the invention is safe, effective, inexpensive, and can be produced on a large scale.Fig. 1. Comparison of peptide-protein conjugation efficiency for various carrier proteins. A. Conjugation with the BSA protein allowed for conjugation with 3 peptide molecules. B. Conjugation with the HSA protein allowed for conjugation with 35 peptide molecules. C. Conjugation with the DT protein allowed for conjugation with 2 peptide molecules. D. Conjugation with the TT protein allowed for conjugation with 6 peptide molecules.

[0024] Fig. 2. Preparation of the GLNRYDERYIGGGGGGC-HSA conjugate. A. Preparation of the carrier protein for conjugation by purification of the HSA monomer using gel filtration chromatography on a Superdex S200. B. Homogeneous monomeric preparation of HSA protein purified on a Superdex S200 column. C. MALDI-TOF-MS spectrum allowing for the mass determination of the carrier protein, the protein after bromoacetylation, and the protein-peptide conjugate. Peptide-protein conjugate: GLNRYDERYIGGGGGGC-HSA (MWavg = 123162.847 Da), the carrier protein after bromoacetylation (MWavg = 71714.226 Da), and the carrier protein before bromoacetylation (HSA, MWavg = 66620.325 Da). D. Confirmation of the presence and purity of the HSA-carrier protein conjugate conjugated with 29 peptides: GLNRYDERYIGGGGGGC-HSA in an SDS-PAGE polyacrylamide gel. E. Immunoreactivity of OmpC protein - a natural antigen from S. flexneri (positive control), and with the GLNRYDERYIGGGGGGC-HSA conjugate with 29 peptides. The negative control, HSA protein, showed no reactivity with the human cord blood antibodies used in the tests.

[0025] Fig. 3. Cytotoxicity assay results. Effect of the GLNRYDERYIGGGGGGC-HSA conjugate, OmpC protein, HSA carrier protein, and free GLNRYDERYIGGGGGGC peptide on K562 cell viability in vitro.

[0026] Fig. 4. Reactivity of HSA conjugates with the GLNRYDERYIGC peptide and the GGGGG peptide with the sequence GLNRYDERYIGGGGGGC with antibodies from human cord blood. The reactivity of HSA:GLNRYDERYIGGGGGGC and HSA:GLNRYDERYIGC conjugates with antibodies from human cord blood was tested in a dot-blot assay. The antibodies from human cord blood showed more intense reactivity with the HSA protein conjugates and the peptide containing the glycine linker. No reactivity with HSA protein (negative control) was observed, indicating high immunoreactivity of the human cord blood antibodies with the GLNRYDERYIG epitope.

[0027] Figure 5. Reactivity of HSA conjugates with the peptide GLNRYDERYIGC and a peptide with a GGGGG linker of the sequence GLNRYDERYIGGGGGGC with the anti-RYDERY monoclonal antibody.Figure 6. Reactivity of the carrier proteins DT, TT, BSA, and HSA, and of DT, TT, BSA, and HSA conjugates with the peptide with a GGGGG linker of the sequence GLNRYDERYIGGGGGGC with the anti-RYDERY monoclonal antibody.

[0028] Figure 7. Reactivity of HSA and HSA conjugates with the peptide with a GGGGG linker of the sequence GLNRYDERYIGGGGGGC of various loadings with antibodies from human cord blood.Examples

[0029] Example 1. Selection of a carrier molecule enabling maximum peptide loading.

[0030] The goal was to prepare a prototype conjugate vaccine based on a carrier protein and a peptide antigen. Several carrier proteins were tested, and the protein selected that allowed conjugation with the largest possible number of peptides constituting the vaccine antigen. The following carrier proteins were tested: TT (tetanus toxoid), DT (dyptheria toxoid), BSA (bovine blood albumin), and HSA (human blood albumin). For conjugation, both a peptide without a glycine linker (GLNRYDERYIGC) and, in subsequent steps, a peptide with a glycine linker (GLNRYDERYIGGGGGGC:SEQ ID NO: 1 ) were used, enabling the peptide antigen to be distanced from the protein carrier and its better exposure after immunization. The peptide was conjugated via the C-terminal residue (Cys, cysteine) of the peptide to a carrier protein molecule bromoacetylated with an N-hydroxybromoacetyl ester (bromoacetylation allows for the formation of an active NH2 residue that can be conjugated with the -SH group of the peptide cysteine). To optimize the protein bromoacetylation and conjugation process, various chemical reaction conditions were tested, such as optimal pH, incubation time, and concentration of carrier proteins and peptides, as well as the addition of reagents preventing peptide dimerization prior to conjugation with the carrier (trimethylphosphine was tested).

[0031] The final protocol for preparing the protein-peptide conjugate included the following steps:

[0032] 1. Purification of the carrier proteins (TT, DT, BSA, and HSA) using sieve chromatography in carbonate buffer, pH 8.3. The proteins were then monitored for purity and the recovery of the monomeric form of each protein in a polyacrylamide gel.

[0033] 2. Bromoacetylation was carried out in 0.2 M carbonate buffer, pH 8.3, using bromoacetyl acid N-hydroxysuccinate ester at a concentration of 1 mg of ester per 1 mg of protein (maintaining a protein concentration of c = 2 mg / ml) for 3 hours at room temperature on a rotary mixer.

[0034] 3. The bromoacetylated carrier protein was purified from excess ester on a 30 kDa membrane. Additionally, the carrier protein was washed with 4 volumes of carbonate buffer with 2 mM EDTA, pH 8.3, to a concentration in the range of 10-20 mg / ml. The degree of bromoacetylation was monitored using a MALDI-TOF-MS spectrometer. The possibility of increasing the degree of bromoacetylation of carrier proteins by prior cationization of the protein with ethylenediamine was also tested, which should increase the number of -NH3 groups on the protein and thus improve bromoacetylation - no significant improvement was observed and this step was abandoned.

[0035] 4. Peptides containing thiol groups for conjugation were suspended in 0.2 M carbonate buffer with 2 mM EDTA, pH 8.3, at a concentration of 10-50 mg / ml. Test experiments did not demonstrate improved conjugation using trimethylphosphine -this reagent actuallyhindered the subsequent conjugation process and was therefore excluded from the protocol.

[0036] 5. Bromoacetylated protein, pH 8.5, was added to the peptide solution within 1 minute. The conjugation reaction was carried out overnight, approximately 16 hours, at room temperature on a rotary mixer in a sealed vessel under argon. Unreacted bromoacetyl groups on the carrier protein molecule were deactivated using 10 pl of 2-mercaptoethanol per ml of reaction mixture during incubation for 1 hour at room temperature. The degree of peptide occupancy on the carrier protein was monitored on a MALDI-TOF-MS spectrometer.

[0037] As a result of the analyses and conjugation, the HSA protein was selected for further stages of the project as the carrier for conjugation with the peptide GLNRYDERYIGGGGGGC, which showed the best peptide occupancy after conjugation (Fig. 1 ). The selection was based on the following criteria:

[0038] 1. The HSA protein showed the best degree of peptide occupancy (35 peptide molecules), while the remaining molecules were successfully conjugated with fewer peptide molecules: TT - 6 peptide molecules, DT - 2 peptide molecules, and BSA - 3 peptide molecules.

[0039] 2. The HSA protein showed a high degree of stability in aqueous solution during variable conditions during bromoacetylation and conjugation (DT and TT proteins precipitated from solution when pH changed during bromoacetylation, leading to their denaturation and significant losses in the resulting preparation).

[0040] 3. The HSA protein is a much cheaper carrier (100 pg - EUR 25) than, for example, toxoids, whose production and purification process is much more labor-intensive (e.g., the cost of TT is EUR 2000 per 100 pg) - therefore, this will be of great importance in the case of potential implementation and production of the vaccine on an industrial scale.

[0041] 4. The HSA protein is human blood albumin -usingthis protein as a carrierforthe peptide antigen should not affect the development of an additional immune response directed at the vaccine carrier. The complete process for obtaining the GLNRYDERYIGGGGGGC-HSA conjugate is shown in Fig.2.

[0042] As a result of optimizing the conjugation method, a prototype vaccine based on an immunogenic peptide (GLNRYDERYIGGGGGGC) and the HSA carrier protein was obtained with very good conjugation reaction efficiency, allowing for an average loading of 29 peptide molecules per HSA molecule.

[0043] Therefore, the use of the GLNRYDERYIGGGGGGC-HSA vaccine prototype offers real opportunities for improved immunogenic properties, improved vaccine stability, reduced price, and minimized side effects.Example 2. Testing the cytotoxic properties of the vaccine prototype.

[0044] The aim of the experiment was to determine whether the GLNRYDERYIGGGGGGC-HSA conjugate exhibited toxic properties towards human K562 cells. For this purpose, cytotoxicity tests were performed in vitro on culture plates.

[0045] K562 cells derived from cell culture were distributed in the wells of a 96-well plate at a density of 4,000 cells / well. The cells were then incubated for 24 hours in a cell culture incubator (37°C, 5% CO2) and exposed to antigens in a series of dilutions for another 72 hours. After this time, cell viability was checked using AlamarBlue, which allows for the determination of the metabolic activity of cells in culture and, therefore, their viability after contact with the test substance. As part of this task, control components (negative controls: the GLNRYDERYIGGGGGGC peptide and the HSA carrier protein) were also tested, as well as the OmpC protein itself, which contains the epitope we studied and which had previously demonstrated immunogenic properties. Example 2 demonstrated the lack of toxicity of the GLNRYDERYIGGGGGGC-HSA conjugate at concentrations below 1 pM (such a molecule is typically considered non-toxic). Furthermore, the conjugate did not exhibit cytotoxic properties up to 10 pM, and its toxic properties became apparent only after exceeding 30 pM (~IC50), reaching high toxicity at 100 pM (>IC90). It should be noted that the cytotoxic properties of the conjugate are much lower than those of the OmpC protein itself at the same concentrations.

[0046] Example 3. Examination of the antigenic properties of the obtained conjugates with cord blood sera.

[0047] The aim of the experiment was to examine the antibody reactivity of cord blood sera from various patients with the obtained conjugates containing a peptide without a linker and a peptide with a linker.

[0048] The dot-blot procedure was performed according to the protocol:

[0049] 1. The Immobilon P membrane was activated in methanol and then rinsed in TBS-T. The membrane was allowed to dry.

[0050] 2.2 pl of methanol was applied to the separated zones of the membrane, followed by the prepared samples with antigen.

[0051] 3. The membrane was blocked with 1% BSA (Thermo Scientific) in TBS-T on a laboratory cradle at room temperature for 1 hour.4. After the incubation period, the membrane was washed three times with TBS-T buffer. After each addition of TBS-T buffer, the membrane was incubated for 15 minutes, 5 minutes, and 5 minutes, respectively.5. Antigen reactivity with antibodies present in cord blood serum (H1 and H7 serum) was tested. The serum sample was diluted 1:100 in 1% BSA in TBS-T buffer. The membrane was incubated on a laboratory rocker at room temperature for 1 hour.

[0052] 6. After the incubation period, the membrane was washed three times with TBS-T buffer. After each addition of TBS-T buffer, the membrane was incubated for 15 minutes, 5 minutes, and 5 minutes, respectively.

[0053] 7. After washing, a reaction with a secondary antibody (1:10,000) was performed. Antihuman IgG antibody with alkaline phosphatase (Promega) was used for the reaction. The membrane was incubated on a laboratory rocker at room temperature for 1 hour.

[0054] 8. After the incubation period, the membrane was washed three times with TBS-T buffer. After each addition of TBS-T buffer, the membrane was incubated for 15 minutes, 5 minutes, and 5 minutes, respectively.

[0055] 9. The dot blots were developed using a solution containing phosphatase substrate for 1 minute and then placed in MilliQ water.

[0056] Example 4. Testing the specificity of the obtained conjugates with the anti-OmpC epitope monoclonal antibody (RYDERY)

[0057] The Western blot procedure was performed according to the protocol:

[0058] 1. Antigens (OmpC, HSA-GLNRYDERYIGGGGGGC conjugate and HSAGLNRYDERYIGC, as well as the HSA carrier protein) were separated in a 12.5% polyacrylamide gel under denaturing conditions (SDS-PAGE).

[0059] 2. Antigens were transferred (100 V, 1 h) to an Immobilon P membrane.

[0060] 3. The membrane was blocked with 1% BSA in TBS-T (stock solution of 10% BSA in PBS from Thermo Scientific) on a laboratory rocker at room temperature for 1 hour.4. After the incubation period, the membrane was washed three times using TBS-T buffer. After each addition of TBS-T buffer, the membrane was incubated for 3 x 5 minutes.

[0061] 5. Antigen reactivity was checked with the anti-RYDERY monoclonal antibody diluted 1 :100 in 1 % BSA in TBS-T buffer. The membrane was incubated on a laboratory rocker at room temperature for 1 hour.

[0062] 6. After the incubation period, the membrane was washed three times using TBS-T buffer. After each addition of TBS-T buffer, the membrane was incubated for 3 x 5 minutes.

[0063] 7. After washing, a reaction was performed with a secondary antibody (1:10,000). Antimouse IgG antibody with alkaline phosphatase (Promega) was used for the reaction. 8. The membrane was incubated on a laboratory rocker at room temperature for 1 hour.9. After the incubation period, the membrane was washed three times using TBS-T buffer. After each addition of TBS-T buffer, the membrane was incubated for 3 x 5 minutes.

[0064] 10. The prepared blots were developed using a solution containing phosphatase substrate for 1 minute and then placed in MiliQ water.

[0065] The experiment demonstrated very good reactivity with OmpC antigens (positive control) as well as conjugates containing the RYDERY epitope sequence of the OmpC protein. However, no reactivity with the HSA carrier protein was observed.

[0066] Example 5. Testing the specificity of the obtained conjugates with the anti-OmpC epitope monoclonal antibody (RYDERY) in an ELISA.

[0067] The ELISA procedure was performed according to the guidelines:

[0068] 1) Coating the plate with antigen solution. A 96-well plate (MaxiSorp) was used. The antigen to be analyzed (1 pg / 100 pl / well) was applied to the plate: carrier proteins, and carrier protein conjugates with the GLNRYDERYIGGGGGGC peptide. The plate was coated for 1 hour at room temperature and then overnight in a refrigerator at 4°C. After coating, excess antigen was removed and washed.

[0069] 2) Blocking - The wells were blocked with 3% PVA in TBS-T: 250 pl / well. Blocked for 1 hour at room temperature.

[0070] 3) Washing - Plates were washed with TBS-T. The wells were washed with 3x250 pl / well.

[0071] 4) Incubation with primary antibody - To determine the reactivity of IgG antibodies with the antigen, an anti-RYDERY monoclonal antibody was used at a dilution of 1:5,000. Incubation was carried out for 1 hour at room temperature.

[0072] 5) Washing - Plates were washed with TBS-T. The plates were washed with 3x250 pl / well.

[0073] 6) Incubation with secondary antibody -To determine the reactivity, an anti-mouse IgG antibody conjugated with alkaline phosphatase (dilution 1:10,0000) was used at 100 pl / well. Incubation was carried out for 1 hour at room temperature. 7) Washing - Plates were washed with TBS-T. The wells were washed 3x250 pl / well.

[0074] 8) Colorimetric reaction - to quantify the level of antibodies against the analyzed antigens in cord blood serum, 200 pl / well of AP Yellow phosphatase substrate (pNPP, Merck) was used. The plate was incubated for 30 minutes. The reaction was stopped by adding 50 pl / well of 3M NaOH. Absorbance was read at 405 nm.

[0075] The reactivity of the conjugates and their carrier proteins was compared. It was shown that the monoclonal antibody recognizing the RYDERY epitope exhibited high affinity forthe peptides constituting the epitope sequence compared to their carrier proteins. Additionally, the degree of reactivity of monoclonal antibodies was found to be highest for the HSA conjugate: GLNRYDERYIGGGGGGC, which was best covered with peptide molecules (29 peptide molecules / 1 HSA molecule).

[0076] Example 6. Examination of the effect of peptide loading on the carrier protein on the reactivity of the conjugate with cord blood antibodies.

[0077] Dot-Blot Procedure:

[0078] 1. The Immobilon P membrane was activated in methanol and then rinsed in TBST. The membrane was allowed to dry.

[0079] 2. 1 pl of methanol was applied to the separated zones of the membrane, followed by the prepared antigen samples.

[0080] 3. The membrane was blocked with 1% BSA (Thermo Scientific) in TBS-T on a laboratory rocker at room temperature for 1 hour.

[0081] 4. After the incubation period, the membrane was washed three times with TBS-T buffer. After each addition of TBS-T buffer, the membrane was incubated for 15 minutes, 5 minutes, and 5 minutes, respectively.

[0082] 5. After washing, the antigens were tested for reactivity with the primary antibody present in cord blood serum (H7 serum). The serum sample was diluted 1:100 in 1% BSA in TBS-T buffer. The membrane was incubated on a laboratory rocker at room temperature for 1 hour.

[0083] 6. After the incubation period, the membrane was washed three times with TBS-T buffer. After each addition of TBS-T buffer, the membrane was incubated for 15 minutes, 5 minutes, and 5 minutes, respectively.

[0084] 7. A reaction was performed with the secondary antibody (1:10,000). Anti-human IgG antibody with alkaline phosphatase (Promega) was used for the reaction. The membrane was incubated on a laboratory rocker at room temperature for 1 hour.

[0085] 8. After the incubation period, the membrane was washed three times using TBS-T buffer. After each addition of TBS-T buffer, the membrane was incubated for 15 minutes, 5 minutes, and 5 minutes, respectively.

[0086] 9. The prepared blots were developed using a solution containing phosphatase substrate for 1 minute and then placed in MiliQ water.

[0087] The degree of loading of the HSA carrier protein affected the reactivity of antibodies present in human cord blood was examined. No reactivity with the carrier protein was observed, and an increase in the intensity of the reactivity of the HSA: GLNRYDERYIGCconjugates was demonstrated depending on the loading of the carrier protein molecule with 14, 21, and 29 peptide molecules.Sequence list

[0088] <110> Institute of Immunology and Experimental Therapy, Polish Academy of Sciences <120 Conjugate containing an antigenic peptide and its use for preventing bacterial infections caused by Shigella

[0089] <130 PK / 11658 / AR

[0090] <160 1

[0091] <170 Patentin version 3.5

[0092] <210> 1

[0093] <211> 17

[0094] <212> PRT

[0095] <213> artificial

[0096] <400 1

[0097] Gly Leu Asn Arg Tyr Asp Glu Arg Tyr He Gly Gly Gly Gly Gly Gly

[0098] 1 5 10 15

[0099] Cys

Claims

Claims1. A conjugate composed of an antigenic peptide presenting an epitope of the Shigella flexneri 3a OmpC protein of SEQ ID NO: 1, conjugated via the C-terminal residue of the peptide to a human serum albumin (HSA) carrier protein molecule.

2. A vaccine composition comprising the conjugate of claim 1 and an adjuvant.

3. A conjugate of claim 1 for use in preventing bacterial infections caused by Shigella bacteria.

4. A conjugate for use according to claim 3, characterized in that the bacterial infection is bacillary dysentery.

Images